Growth Hormone ("GH") is a peptide having 191 amino acids which stimulates the production of numerous different growth factors, e.g. insulin-like growth factor I (IGF-I) and so promotes growth of numerous tissues (skeleton, connective tissue, muscle and viscera) and physiological activities (raising nucleic acid and protein synthesis and lipolysis, but lowering urea secretion).
Release of GH is under the control of releasing and inhibiting factors secreted by the hypothalamus. The primary releasing factor is growth hormone releasing hormone ("GH-RH"); human growth hormone-releasing hormone ("hGH-RH") is a peptide having 44 amino acids. The novel peptides of the present invention relate to analogues of hGH-RH having only residues 1 through 29 ("hGH-RH(1-29)NH.sub.2 "), i.e., to analogues of the peptide which has the amino acid sequence:
Tyr-Ala-Asp-Ala-Ile.sup.5 -Phe-Thr-Asn-Ser-Tyr.sup.10 -Arg-Lys-Val-Leu-Gly.sup.15 -Gln-Leu-Ser-Ala-Arg.sup.20 -Lys-Leu-Leu-Gln-Asp.sup.25 -Ile-Met-Ser-Arg.sup.29 -NH.sub.2 PA1 R.sup.1 is Tyr or His, PA1 R.sup.2 is D-Arg or D-Cit, PA1 R.sup.5 is Ile or Val, PA1 R.sup.6 is Phe, Nal or Phe(Y), in which Y=F, Cl, Br, PA1 R.sup.8 is Asn, Gln, Ser, Thr, Ala, D-Asn, D-Gln, D-Ser, D-Thr, Abu, D-Abu, or Aib, PA1 R.sup.9 is Arg, Har, Lys, Orn, D-Arg, D-Har, D-Lys, D-Orn, Cit, Nle, Tyr (Me), Ser, Ala or Aib, PA1 R.sup.10 is Tyr or Phe(Y), in which Y=H, F, Cl, Br, or OCH.sub.3, PA1 R.sup.12 is Lys, D-Lys, or Orn, PA1 R.sup.13 is Val or Nle, PA1 R.sup.14 is Leu or Nle, PA1 R.sup.15 is Gly, Ala, Abu, Nle or Gin, PA1 R.sup.16 is Gln or Arg, PA1 R.sup.18 is Ser or Nle, PA1 R.sup.19 is Ala or Abu, PA1 R.sup.21 is Lys or Orn, PA1 R.sup.22 is Leu, Ala or Aib, PA1 R.sup.27 is Met, Leu, Nle, Abu, or D-Arg, PA1 R.sup.28 is Arg, D-Arg, Ser, Asn, Asp, Ala or Abu, PA1 R.sup.29 is Arg, D-Arg, Har or D-Har, and pharmaceutically acceptable salts thereof.
GH has been implicated in several diseases. One disease in which GH is involved is acromegaly, in which excessive levels of GH are present. The abnormally enlarged facial and extremity bones of this disease can be treated by administering a GH-RH antagonist.
Further diseases involving GH are diabetic retinopathy and diabetic nephropathy. The damage to the retina and kidneys respectively in these diseases, believed to be due to GH, results in blindness or reduction in kidney function. This damage can be prevented or slowed by administration of an effective GH-RH antagonist.
However, the main applications of GH-RH antagonists would be in the field of cancer (A. V. Schally et al, in Growth Hormone Secretagogues in Clinical Practice, eds. Bercu, B. B. & Walker, R. F., Dekker, New York, pp. 145-162, 1998). IGF-I and -II are potent mitogens for various cancers. By suppressing GH secretion, GH-RH antagonists decreases the synthesis of IGF-I in the liver and other tissues. GH-RH antagonists also reduce the autocrine and paracrine production of IGF-I and/or IGF-II by various tumors. In several experimental cancers, treatment with antagonists of GH-RH produces a reduction in IGF-I and -II, concomitant to inhibition of tumor growth.
In an effort to intervene in these disease and other conditions, some investigators have attempted to control GH levels by using somatostatin, one inhibitor of GH release. However, somatostatin, if administered alone, does not suppress GH or IGF-I levels to a desired degree. If administered in combination with a GH-RH antagonist, somatostatin would improve suppression of IGF-I levels much better.
Scientists have investigated various modifications of GH-RH to elucidate the relationship of the structure of GH-RH to its activity in an effort to provide synthetic congeners with improved agonistic or antagonistic properties. Thus, it was early established that GH-RH fragment comprising residues 1 to 29, or GH-RH(1-29), is the minimum sequence necessary for biological activity. This fragment retains 50% or more of the potency of native GH-RH.
The first described GH-RH antagonist, [Ac-Tyr.sup.1, D-Arg.sup.2 ]hGH-RH(1-29) NH.sub.2, which is generally termed as the "standard antagonist" in the literature, was found to prevent the activation of rat anterior pituitary adenylate cyclase by hGH-RH(1-29)NH.sub.2. The same peptide blocked the action of GH-RH on its receptors in the pituitary and hypothalamus, and inhibited the pulsatile growth hormone secretion.
A considerable number of patents and articles in the open literature disclose analogs of GH-RH which either act as agonists of GH-RH (i.e. act to stimulate the release of GH) or as antagonists of GH-RH (i.e. act to inhibit the release of GH). Most of these peptides are derived from the GH-RH(1-29) peptide sequence, with specific structural modifications which account for their enhanced agonistic or antagonistic properties.
Thus, U.S. Pat. No. 4,659,693 discloses GH-RH antagonistic analogs which contain certain N,N'-dialkyl-omega-guanidino alpha-amino acyl residues in position 2 of the GH-RH(1-29) sequence.
Published application WO 91/16923 reviews earlier attempts to alter the secondary structure of hGH-RH by modifying its amino acid sequence. These earlier attempts include: replacing Tyr.sup.1, Ala.sup.2, Asp.sup.3 or Asn.sup.8 with their D-isomers; replacing Asn.sup.8 with L- or D-Ser, D-Arg, Asn, Thr, Gln or D-Lys; replacing Ser.sup.9 with Ala to enhance amphiphilicity of the region; and replacing Gly.sup.15 with Ala or Aib. When R.sup.2 in the analogs is D-Arg, and R.sup.8, R.sup.9, and R.sup.15 are substituted as indicated above, antagonistic activity is said to result. These antagonistic peptides are said to be suitable for administration as pharmaceutical compositions to treat conditions associated with excessive levels of GH, e.g., acromegaly.
The antagonistic activity of the hGH-RH analogue "[Ser.sup.9 -.PSI.[CH.sub.2 -NH]-Tyr.sup.10 ]hGH-RH(1-29)" of U.S. Pat. No. 5,084,555 was said to result from the pseudopeptide bond (i.e., a peptide bond reduced to a [CH.sub.2 -NH] linkage) between the R.sup.9 and R.sup.10 residues. However, the antagonistic properties of [Ser.sup.9 -.PSI.[CH.sub.2 -NH]-Tyr.sup.10 ]hGH-RH(1-29)were said to be inferior to the standard antagonist, [N-Ac-Tyr.sup.1, D-Arg.sup.2 ]GH-RH(1-29)-NH.sub.2.
U.S. Pat. No. 5,550,212, and U.S. patent application Ser. No. 08/642,472, assigned to the same assignee as the present application, disclose analogs of hGH-RH(1-29)NH.sub.2 said to have enhanced antagonistic properties and prolonged duration of action. These properties are believed to result from replacement of various amino acids and acylation with aromatic or nonpolar acids at the N-terminus of GH-RH(1-29)NH.sub.2. It is noted that in the above U.S. patent and U.S. patent application, R.sup.9 is always Ser, R.sup.16 is Gln or an amino acid forming a lactarn bridge (i.e. Glu), R.sup.28 is Ser, Asn, Asp, Ala or Abu, and R.sup.29 is Agm, Arg-NH.sub.2, Arg-OH, Cit-NH.sub.2, Cit-OH, Har-NH.sub.2, Har-OH, or an amino acid forming a lactam bridge (i.e. Lys or Orn).